![Figure 6. Abrogation of GVHD in Nrf2−/− allo-T cell recipients is associated with a greater proportion of donor-derived CD4+ Helios+ Tregs. (A-G) Lethally irradiated BALB/c recipients were transplanted with WT B6 TCD BM and 0.5 × 106 B6 WT or Nrf2−/− T cells. Donor CD4+ T-cell subsets in recipient spleens were analyzed on day 7 after HCT. Data represent the mean ± standard deviation. (A) Absolute numbers of WT and Nrf2−/− donor-derived CD4+ Th1 (T-bet+) cells, Th2 (GATA-3+) cells, Th17 (RORγt+) cells, and Tregs (FoxP3+CD25+) (n = 15 per group, combined from 3 independent experiments). (B) Representative flow cytometric dot plots showing the frequency of Tregs within donor CD4+ populations. (C) Representative flow cytometric analysis and bar graphs showing the mean fluorescence intensity (MFI) of intracellular Helios expression within donor-derived Tregs (n = 5 per group, 1 of 2 reproducible experiments). (D) The proportion of donor-derived Tregs expressing Helios (n = 15 per group, combined from 2 independent experiments). (E) Representative flow cytometric analysis and bar graph showing the MFI of intracellular FoxP3 expression within donor-derived Tregs (n = 5 per group, 1 of 3 reproducible experiments). (F) IL-2 levels in the recipient sera were analyzed using Luminex (n = 19 or 20 per group combined from 4 independent experiments). (G) An IL-2 secretion assay was performed to detect IL-2 levels on the donor T-cell surface, and MFI was analyzed using flow cytometry (n = 5 per group, 1 of 2 reproducible experiments). (H) WT CD45.1+ B6 TCD BM plus CD45.2+ WT vs Nrf2−/− T cells were transplanted into lethally irradiated CD45.1+ B6 (syngeneic HCT [syn-HCT]) or BALB/c (Allo-HCT) recipients. Bar graphs show the MFI of CD25 receptor and intracellular Helios expression within donor-derived Tregs. Data represent the mean + standard deviation from 1 of 2 reproducible experiments (n = 5 per group). (I) Lethally irradiated BALB/c recipients transplanted with WT B6 TCD BM alone or with a total of 0.5 × 106 B6 putative Treg (CD4+CD25+)-depleted B6 WT or Nrf2−/− conventional CD4+ and CD8+ T cells (Tcon) were monitored daily for survival. Kaplan-Meier analysis showing combined data from 2 independent experiments. (J) Schematic model of the proposed mechanism by which Nrf2 regulates CD4+ T cells in GVHD. *P < .05, **P < .01, ***P < .005, ****P < .0001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/132/26/10.1182_blood-2017-10-812941/6/blood812941f6.png?Expires=1735837200&Signature=HahXjb2qBnNEU3lsnGg8sLBvNjhY2Tu4evJnAnxdWefjhr14MXXNX-zjJ451V0M~jnHsCNk6DhaTblWdssLG2DFIEff77w2zONPye3-hB0725gQBKxxj6Ply6t221ykEMbfNU~4X0X2kujvmbOUXC2BuQi1jk6elJfiPKwLT1WwB1gE9DZuKEBMy-w8A4tdvmTp9vnQwGSOoPEXOx-Llq1SWaIO9~oVakUianMYIWiWxKRx247SYKuK~m0DN0l8fpUDe-ukTwiW8glbBPvxZgadDx4790D7wIfaHduV43ui7DQbViMJA-lnaafjJ3xwZddJZcNoiI-UyllxMQIAwGg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Abrogation of GVHD in Nrf2−/−allo-T cell recipients is associated with a greater proportion of donor-derived CD4+Helios+Tregs. (A-G) Lethally irradiated BALB/c recipients were transplanted with WT B6 TCD BM and 0.5 × 106 B6 WT or Nrf2−/− T cells. Donor CD4+ T-cell subsets in recipient spleens were analyzed on day 7 after HCT. Data represent the mean ± standard deviation. (A) Absolute numbers of WT and Nrf2−/− donor-derived CD4+ Th1 (T-bet+) cells, Th2 (GATA-3+) cells, Th17 (RORγt+) cells, and Tregs (FoxP3+CD25+) (n = 15 per group, combined from 3 independent experiments). (B) Representative flow cytometric dot plots showing the frequency of Tregs within donor CD4+ populations. (C) Representative flow cytometric analysis and bar graphs showing the mean fluorescence intensity (MFI) of intracellular Helios expression within donor-derived Tregs (n = 5 per group, 1 of 2 reproducible experiments). (D) The proportion of donor-derived Tregs expressing Helios (n = 15 per group, combined from 2 independent experiments). (E) Representative flow cytometric analysis and bar graph showing the MFI of intracellular FoxP3 expression within donor-derived Tregs (n = 5 per group, 1 of 3 reproducible experiments). (F) IL-2 levels in the recipient sera were analyzed using Luminex (n = 19 or 20 per group combined from 4 independent experiments). (G) An IL-2 secretion assay was performed to detect IL-2 levels on the donor T-cell surface, and MFI was analyzed using flow cytometry (n = 5 per group, 1 of 2 reproducible experiments). (H) WT CD45.1+ B6 TCD BM plus CD45.2+ WT vs Nrf2−/− T cells were transplanted into lethally irradiated CD45.1+ B6 (syngeneic HCT [syn-HCT]) or BALB/c (Allo-HCT) recipients. Bar graphs show the MFI of CD25 receptor and intracellular Helios expression within donor-derived Tregs. Data represent the mean + standard deviation from 1 of 2 reproducible experiments (n = 5 per group). (I) Lethally irradiated BALB/c recipients transplanted with WT B6 TCD BM alone or with a total of 0.5 × 106 B6 putative Treg (CD4+CD25+)-depleted B6 WT or Nrf2−/− conventional CD4+ and CD8+ T cells (Tcon) were monitored daily for survival. Kaplan-Meier analysis showing combined data from 2 independent experiments. (J) Schematic model of the proposed mechanism by which Nrf2 regulates CD4+ T cells in GVHD. *P < .05, **P < .01, ***P < .005, ****P < .0001.